PROJECT SUMMARY Assembly of trafficking vesicles plays a key role in many human diseases. More than 50 cytoplasmic proteins work together to build and shape the highly curved vesicular coat through the sequential steps of initiating protein assembly, sensing membrane curvature, and driving membrane vesiculation. Meanwhile hundreds of distinct transmembrane cargo proteins compete for space within the crowded environment of the nascent vesicle. While it is clear that multiple protein components must work simultaneously to execute each step, most of what we know about their molecular mechanisms is premised on studies of individual proteins and domains in isolation from one another. How might individual mechanisms work together in a heterogeneous environment? Can novel mechanisms emerge from the simultaneous action of multiple individual remodeling proteins? Is the ?whole? greater than ?the sum of its parts?? Based on substantial published and preliminary results from the first 3.5 years of this project, our central hypothesis is that assembly of multi- component protein networks can synergistically amplify the contributions of individual proteins to key steps of trafficking vesicle biogenesis. Aim 1 will quantify the role of protein networks in membrane curvature sensing and vesiculation. Recently we have demonstrated that seemingly disparate physical mechanisms can work together synergistically to sense membrane curvature and remodel membrane surfaces. These results support our working hypothesis- assembly of protein networks can functionally and synergistically combine multiple mechanisms of membrane remodeling. To test this hypothesis, we will investigate the collaboration between membrane scaffolding, helix insertion, and protein crowding in membrane curvature sensing and vesiculation in vitro and in live cells. Aim 2 will evaluate the ability of protein phase separation to catalyze coated vesicle assembly. Recently we have made the exciting discovery that Eps15 and FCHo, key nucleators of endocytic vesicles, can assemble into protein droplets at membrane surfaces. Based on these findings, we will use in vitro and live cell experiments to test the working hypothesis that phase-separated protein droplets provide a dynamic platform for catalyzing coated pits by controlling their spatial and temporal assembly. Aim 3 will measure the impact of cargo-cargo interaction networks on endocytic uptake. It is increasingly appreciated that the endocytic uptake of each cargo protein is dependent upon the diverse network of other cargo proteins present at the cell surface. Therefore, we will use model cargo and disease-related receptors to evaluate the working hypothesis that the molecular content of CCPs depends directly on competition and collaboration among cargo molecules. Significantly, this work will explain how diverse molecular mechanisms work together synergistically in trafficking vesicle biogenesis, suggesting the innovative premise that the functional role of each protein can only be understood in the context of the network of other proteins and mechanisms with which it interacts. !